|Title of Invention||
ANTIESTROGENIC ACTIVITY OF PHYTOSTEROLS FROM FENUGREEK SEEDS
|Abstract||Antiestrogenic Phytosterol Extract From Fenugreek Seeds This invention relates to a phytosterol extract from fenugreek seeds which has an antiestrogenic effect on the uterine weights and the female reproductive hormone levels in the rats. The phytosterol extract comprising of y- Sitosterol, and ergo-5-en-3-ol wherein the weight ratio of y-Sitosterol and ergo-5-en-3-ol is in the range of 81:19 to 85:15 when tested in female Albino Wistar rats at the dose rate of 8mg/day for 3 consecutive oestrous cycles demonstrated a highly significant difference in the uterine weights of the rats, such that the treated groups showed a marked drop in the uterine weight as compared to those in the control group. The levels of FSH, LH, progesterone and estrogen in the treated groups were also found to be below those of the control group. In conclusion, the present findings suggest that fenugreek phytosterols are capable of eliciting responses which are consistent with traditional belief that fenugreek can affect reproductive physiology on account of its antiestrogenicity.|
|Full Text||FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
THE PATENTS RULES, 2003
APPLICATION FOR GRANT OF A PATENT
[See section 7,54 and 135, rule 20 (1)]
1. TITLE OF THE INVENTION: Antiestrogenic activity of Phytosterols from Fenugreek seeds
(a) Name : Amin Jyoti
(b) Nationality: Indian
(c) Address : C/201 Akruti Aneri, Behind Marol Maroshi Bus Depot, Marol, Andheri (E), Mumbai -400 059, Maharashtra. India.
(a) Name : Mandalika Subhadra
(b) Nationality: Indian
(c) Address: Department of Foods, Nutrition and Dietetics, College Of Home Science, Nirmala Niketan, 49 New Marine Lines, Mumbai-400 020, Maharashtra, India.
(a) Name : Variyar Prasad
(b) Nationality: Indian
(c) Address: Food, Flavor & Aroma Chemistry Section, Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085, Maharashtra, India.
(a) Name : Chatterjee Suchandra
(b) Nationality: Indian
(c) Address: Food, Flavor & Aroma Chemistry Section, Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai -400 085, Maharashtra, India.
(a) Name : Subbulakshmi Gurumurthy
(b) Nationality: India
(c) Address: 705, Ananddhaml, Society Road, Opposite Amboli Pathak, Andheri (E), Mumbai -400 059, Maharashtra. India.
(a) Name : Sharma Arun
(b) Nationality: Indian
(c) Address: Head, Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION COMPLETE
The following specification particularly describes the invention and manner in which it is to be performed
19 MAR 2008
FIELD OF INVENTION:
This invention relates to a phytosterol extract from fenugreek seeds having effective antiestrogenic activity. In the pharmaceutical industry these phytosterols are suitable for therapeutic applications where estrogen antagonistic behavior is sought, for example- post partum to regain the uterus size and could be extrapolated to the possibility of female contraception.
BACKGROUND OF INVENTION
Phytosterols are phytochemicals naturally occurring in plants. More than 40 phytosterols have been traced till date, of which beta-sitosterol is the predominant plant sterol comprising nearly about 50% of dietary phytosterols followed by campesterol (about 33%) and stigmasterol (about 2 to 5%). brassicasterol, delta-7-stigmasterol and delta-7-avenasterol. These compounds have a steroidal nucleus i.e. cyclopentanoper- hydrophenanthrene ring with 3 beta hydroxyl substitution and a 5-6 double bond (Mellies M.J. et al 1976). They are usually 28-29 carbon compounds with 1 or 2 double bonds, (one typically in the steroid nucleus and the other in the side chain).
The dietary intake of phytosterols varies from 167-437 mg/day with their concentrations highest in the vegetarian diets but their absorption is in trace amounts. Figs, corn, peas, rice bran, leafy vegetables like amaranth, sesame seed, wheat germ, pistachios, pumpkin seed kernel and oils from walnuts, almonds, peanuts, hazelnuts, soybean sea buckthorn are good sources of phytosterols.
The benefits of phytosterols have been associated with the management of hypercholesterolemia and cancer and their intake either in the form of supplements or additives have become increasingly popular.
Role of phytosterols in the reproductive physiology
■ A phytosterol mixture (5mg/kg/day) when fed to mice led to increased plasma levels of testosterone in the third & fifth generation of male pups and increased testicular levels of testosterone in the third generation. A decrease in the relative uterine weights in the third and fifth generation of the female pups and an increase of plasma estradiol levels was also observed in the fourth generation. Despite these changes, phytosterol exposure did
not lead to any harmful effects on the reproduction of the mice (Ryokkynen A. Kayhko U.R, et al 2005).
■ Beta-sitosterol can be used as a vaginal contraceptive as it inhibited sperm motility. The n-butanol fraction of Alstonia macrophylla leaves at a dose of 100 ug/ml demonstrated a marked inhibition of sperm motility (nearly 50-60% of the goat spermatozoa lost their motility) and at a dose of 400 ug/ml showed complete inhibition of sperm forward motility at 0 min. This n butanol fraction contained beta-sitosterol (Chattopadhyay D., et al 2005).
■ The perinatal exposure of beta-sitosterol (50 mg/kg/day) caused alterations in the weight of the reproductive organs of mink (Mustela vison). The parent generation was exposed for more than 9 months to beta sitosterol while the next generation was exposed via gestation and lactation. The phytosterol exposure led to an increase in the relative testicular and prostate weight in the male offspring of the test group and a drop in the relative uterine weights of the female offspring (Ryokkynen A, et al 2005).
■ The methanolic extract of the roots of Rumex steudelii containing phytosterols was investigated for their antifertility activity in female rats at a dose of 5 g/kg in mice. A significant (p Phytosterol supplementation should be avoided in pregnancy, lactation and genetic disorders like sitosterolemia and cerebrotendinotic xanthomatosis (http://www.PDRhealth.com).
Thus, phytosterols (especially beta sitosterol) are shown to affect the reproductive physiology. Similarly, there has been a folkloric reputation of certain foods that induce fertility where as certain others that have the opposite effect. For example, Pomegranate (Ananas comosus) was a symbol of fertility in the orient religions and Greek mythology. In Egypt the pollen grains of date palm (Phoenix dactylifera) was used to induce fertility in women whereas moghat (Clossostemon brugieri) was used as a post partum beverage (Farnsworth et al, 1975). Self-induced abortionists are reported to have tried tea infusions of hot spices like oregano,
cinnamon or oregano mixed with chocolate (Misago & Fonseca, 1999). The practice of taking ginger, clove and combination of ergot, gin and vinegar to induce bleeding and effect abortion is also common. In Nigeria women consumed potash mixed with lime juice, thrice a day for 3 days to terminate a month old conception, but for pregnancies older than a month cactus juice with fresh egg is used. To effect abortion after 2 months of pregnancy, a stronger solution of potash, lime juice, washing blue with a generous helping of gin is used. In India women are known to have jaggery with omum (ajwain) along with generous doses of ghee with the belief that the heat giving effect of the mixture would cause stomach cramps and disturb conception. Raw papaya, cooked banana stem, pineapple, mango are supposed to interfere with pregnancy (Mankekar, 1973). The antifertility effect of certain spices and condiments has been researched earlier as shown below.
Cumin (Cuminum cyminum) Seeds - Anti implantation activity -> aqueous & alcoholic extracts at 100-200mg/kgB.wt. (Garg S.K., 1976)
Celery (Apium graveolens) Seeds - Uterotonic activity (Saxena V.K.,1973)
Mint (Mentha arvensis) Leaves - Anti implantation activity at 100-500mg/kgB.wt. (Bodhankar et al, 1974)
Feeding diets containing 30% fenugreek seeds to male and female white New Zealand rabbits caused a reduction in the testis weight, the plasma concentration of the androgen hormone and sperm concentrations in the treated males. It also led to a reduction of developing fetuses and of the litter size but an increase the circulating plasma progesterone concentrations in females (Kassem A., et al., 2006) As seen in the above table an air of mystery still revolves around most of the food substances and the active components present in them that claimed to have estrogenic or antiestrogenic properties and these still remain unexplored and unexploited. In view of this of 90 elderly women were surveyed to collect information on the emmenagogic, uterotonic or abortifacient foods consumed in various Hindu communities. Fenugreek seeds were one amongst the commonly reported spices by the participants, as a food item used for the above-mentioned purpose. It was a common belief that fenugreek acts as a galactogogue, but some women were of
the opinion that its consumption also helped to expel the residual products of delivery from the womb post partum. Fenugreek has been known for its effects as an appetizer (Petit PR,' et aI1995) hypocholesterolemic (Sowmya P., and Rajyalakshmi P. 1999), immunostimulatory (Bin-Hafeez B. et al 2003) and hypoglycemic (Bhatia K., et al 2006) agent. It is also proposed to be a galactagogue (Damanik R., Wahlqvist M.L., Wattanapenpaiboon N. 2004, Gabay M.P., 2002) and an aphrodisiac (Kamhi E., 2004).
Based on the above mentioned information, the need for scientifically testing the effects of fenugreek on the parameters of reproductive physiology that are influenced by estrogen has been felt by the claimants.
The term "emmenagogic" as used herein means a substance that can induce menstruation.
The term "uterotonic " as used herein means a substance that can increase the tonus of the uterine
The term "abortifacient" as used herein means a substance that can cause an abortion.
The term" galactagogue" as used herein means a substance that can promote the flow of milk.
OBJECT OF INVENTION
Accordingly, it is an object of this invention to provide an antiestrogenic phytosterol extract from
fenugreek seeds, which is effective to regain the uterus size post partum and could be further
extrapolated to the possibility of female contraception.
Upon further study of the specification and appended claims, further objects and advantages of
this invention will become apparent to those skilled in the art.
This object has been attained by the present invention by providing phytosterol extract of y-
Sitosterol and ergo-5-en-3-ol wherein the weight ratio of y- Sitosterol and ergo-5-en-3-ol is in the
range of 81:19 to 85:15.
STATEMENT OF INVENTION
Accordingly the invention provides a phytosterol extract comprising of y- Sitosterol and ergo-5-
en-3-ol in the weight ratio of about 81:19 to 85:15 isolated from fenugreek seeds for antiestrogenic activity.
In order to obtain a phytosterol extract comprising of y- Sitosterol and ergo-5-en-3-ol, a phytosterol extract, hexane extract was isolated from fenugreek seeds by Soxhlet extraction method. This hexane extract was subjected to preparative thin layer chromatography (TLC) in a chloroform : diethyl ether (90:10) solvent system. A gross sterol band thus eluted was then purified by multiple column chromatographies, medium pressure liquid chromatography using eluting solvents such as hexane: chloroform 90:10, 85:15, 80:20, 70:30 and methanol in a sequential manner. Finer purification was done by repeated preparative thin layer chromatography in the reverse phase. Finally identification was done using GC-MS (EI, 70eV), m/z (rel. inten.%) coupled with an 'H N.M.R and IR assay done using a Bruker 300 MHz FTNMR Spectrophotometer (CDC13, TMS) and a Nicolet 430 FTlR Spectrophotometer (KBr)
Analytical results demonstrated the presence of y- Sitosterol as the major sterol in the phytosterol
fraction obtained from the hexane extract of fenugreek.
'H NMR Spectrum (200 MHz. CDCl3, TMS): Signals at 8- 0.68 (3H, s, C-18), 0.80 (3H, d, C-27
overlapped), 0.82 (3H, d, C-26 overlapped), 0.84 (3H, t, C-29), 0.92 (3H, d, C-21) and 1.01 (3H,
s, C-19) accounted for six methyl groups. A multiplet at 6- 3.51 (1H) showed a hydroxyi group.
A characteristic signal at 5- 5.34 (1H, d) indicated an olefinic proton.
MS (EI. 70eV).m/z (rel. inten.%): 414 (6), 145 (45), 95 (100), 83 (26), 93 (85), 71 (46), 55 (27).
IR (KBr): 3425, 2960, 2940, 2868, 1640, 1465, 1385, 1055, 1030, 970, 810 cm1.
The second important compound was ergo-5-en-3-ol in the phytosterol extract, but it was present in a small amount.
BRIEF DESCRIPTION OF THE DRAWING:
The structures of the two major phytosterols in the extract i.e.y- Sitosterol and ergo-5-en-3-ol are provided.
FIG. 1 shows the structure of y- Sitosterol. FIG.2 shows the structure of ergo-5-en-3-ol.
The examples below are used for a more detailed explanation of this invention. Example 1
1500mg of the hexane extract was subjected to preparative TLC in a chloroform : diethyl ether (90:10) solvent system from which a sterol band was eluted. The isolated band was further subjected to saponification followed by acetylation overnight using distilled pyridine and acetic anhydride (1:2). The saponified acetylated residue was finally injected into the GC- MS for tentative identification. A ratio of y- Sitosterol to ergo-5-en-3-ol of about 85: 15 were obtained in the GC-MS profile.
1600mg of the hexane extract was subjected to preparative and analytical TLC, followed by saponification and acetylation. Preparative TLC using petroleum ether : diethyl ether (90:10) solvent system was carried out with the saponified acetylated extract. A sterol band thus eluted was subjected to repeated reverse phase preparative TLC to yield an extract containing a ratio of y- Sitosterol to ergo-5-en-3-ol of about 81: 19 as seen in the GC- MS profile.
7.4g and 19.5g of hexane extract was subjected to column chromatography and medium pressure
liquid chromatography using eluting solvents such as hexane: chloroform 90:10, 85:15, 80:20,
70:30 and methanol in a sequential manner, further purified by multiple preparative TLC and
finally subjected to GC- MS for tentative identification. A ratio of y- Sitosterol to ergo-5-en-3-ol
of about 84: 16 was obtained in the phytosterol extract thus obtained.
The ratio of y- Sitosterol and ergo-5-en-3-ol was in the range of 81:19 to 85:15
Further more, the antiestrogenic activity of the phytosterol extract comprising of y- Sitosterol, and ergo-5-en-3-ol was measured in vivo by its effect on the uterine weights and the reproductive hormone levels in rats.
The uterus is a classical target organ to evaluate estrogenic activity as the function of the uterus is regulated by the female sex hormones (Diel et al 2000). Adult female Albino Wistar rats (body weight= 150-200g) were dosed with the hexane extract and phytosterol extract orally once at a dose rate of 8mg/day for 3 consecutive oestrous cycles. A control group was also maintained and it was given distilled water as sham treatment. To monitor the phase of the estrous cycle, vaginal smears were taken daily and the smears were subjected to staining. All animals were sacrificed at the end of each of three estrous cycles and blood samples collected by cardiac puncture were analyzed for reproductive hormones such as estrogen, progesterone, follicular stimulating hormone (FSH) and luteinizing hormone (LH). After sacrifice, the uteri were rapidly removed, freed from fat and connective tissue and weighted. On being fed to the rats orally, both the extracts did not affect the food and water intake of the rats. After 12 days there was no significant difference in the increase in the body weights of the control (10.83g), hexane extract group (8.33g) and phytosterol extract group (9.17g) of rats. However the study demonstrated a highly significant difference (p Table 1: Effect Of Fenugreek Extracts On The Uterine : Body Weight Ratio Of Rats:
As seen in the Table No.l a highly significant difference was seen in the mean uterine : body weight ratio of the control, hexane extract group and the phytosterol extract group respectively. A decrease in their uterine weights in the hexane and phytosterol fed groups indicates the antiestrogenic effect of these extracts on the uterus.
Literature demonstrates that on feeding diets containing 30% fenugreek seeds, a decrease in the testis weight, histopathologicai damage to the seminiferous tubules and interstitial tissues; and a decrease in the plasma concentration of the androgen hormone and sperm concentrations
was observed in male rabbits. In the females, the reduction of both fetal, placental weights at 20 days of gestation and of the litter size indicated a significant reduction of developing fetuses (Kassem et al 2006). Perinatal exposure of beta-sitosterol at a dose of 50 mg/kg/day led to alterations in the weight of the reproductive organs of mink (Mustek vison). Ryokkynen, Nieminen et al (2005) demonstrated that beta sitosterol exposure of the parent generation for >9 months and of the next generation via gestation and lactation, caused an increase in the relative testicular and prostate weight in the male offspring of the test group and a decrease in the relative uterine weights of the female offspring. In vivo studies also suggested that beta-sitosterol produces a weak estrogenic effect only at a low dose (6.2 micrograms/dl), but is unable to maintain such an effect at higher doses (Rosenblum et al 1993).
Chattopadhyay et al (2005) demonstrated the use of beta-sitosterol as a vaginal contraceptive as it inhibited sperm motility. They also reported that the n-butanol fraction of Alstonia macrophylla leaves which contained beta-sitosterol when given at a dose of 100 fig/ml exhibited a marked inhibition of sperm motility (nearly 50-60% of the goat spermatozoa lost their motility) and at a dose of 400 ug/ml showed complete inhibition of sperm forward motility at 0 min. Setty et al (1976) also reported that a 2% concentration of the saponin fraction of fenugreek showed invitro spermicidal action against rat and human semen. Multigenerational exposure to phytosterols (5mg/kg/day) in mice led to decrease in the relative uterine weights in the third and fifth generation of the female pups and an increase of plasma estradiol levels was also observed in the fourth generation. Despite these changes, phytosterol exposure did not lead to any harmful effects on the reproduction of the mice (Ryokkynen, Kayhko et al 2005).
Given the premise that, mammalian reproduction involves the interaction of various hormones which orchestrate the functioning of the sex organs, in the present trial serum hormone analysis of the rats was considered imperative.
In the female reproductive system, normally, Gonadrotropin releasing hormone (GnRH), a releasing factor secreted by the hypothalamus stimulates the pituitary to secrete Luteinizing and Follicle stimulating hormone (LH and FSH) which stimulate the gonads to mature and release sex hormones like estrogen, progesterone and testosterone (Tortora and Grabowski 1996). Estrogen, the main female reproductive hormone promotes the growth and maturation of vagina, uterus, fallopian tubes and enlargement of the breast during puberty (Joshi 1986). In non
-pregnant women estrogen with progesterone regulates the normal menstrual cycle by inducing ovulation and by preparing the uterine endometrium to support a conceptus. During pregnancy, estrogen which promotes the development and maintenance of endometrium of the uterus, the uteroplacental blood flow, prepares mammary glands for lactation, physiologically prepares the mother's body for parturition and inhibits release of GnRH, FSH and LH (Bennett 1983). Progesterone acts synergistically with the estrogen to prepare endometrium for implantation of fertilized ovum and mammary glands for milk secretion (Tortora and Grabowski 1996). During pregnancy, progesterone suppresses the contractility of uterine musculature and the immunological rejection of the developing fetus (Gringauz 1997).
In the menstrual cycle, during the follicular phase, a decrease in the levels of estrogen and progesterone causes bleeding due to the shedding of the endometrium while the pituitary gland increases slightly its production of FSH which then stimulates the growth of 3 to 30 follicles (Tortora and Grabowski 1996). The ovulatory phase starts with a very small increase in the FSH levels and an increase in LH which stimulates the dominant follicle to bulge from the surface of the ovary and finally rupture releasing the egg (ovulation). The estrogen level also peaks during the surge and the progesterone level starts to increase (Rosenblatt 2008). The luteal phase follows ovulation and unless fertilization occurs, the LH causes the ruptured follicle to form a corpus luteum, which produces progesterone that causes the endometrium to thicken in preparation for a potential fetus. Later in this phase, the level of estrogen increases and along with progesterone causes the lining of the uterus to thicken more and also increases the milk ducts in the breasts. During the luteal phase, levels of LH and FSH decrease. If the egg is not fertilized, the corpus luteum degenerates. This is followed by a decrease in the progesterone and estrogen levels, which causes the endometrium to degenerate and a new menstrual cycle begins. If the egg is fertilized, the cells around the developing embryo begin to produce a hormone called human chorionic gonadotropin which maintains the corpus luteum, which continues to produce progesterone, until the growing fetus can produce its own hormones (Tortora and Grabowski 1996).
The levels of FSH, LH and estrogen in the treated groups were also found to be below those of the control group (Table No. 2) while the serum progesterone content decreased markedly in the hexane extract fed group but increased in the phytosterol fed group. A deviation from the hormone levels of the control rats observed in the trial, implies alterations in the normal menstrual cycle of the rats when treated with hexane or phytosterol extract. The increase seen in the progesterone seen in the phystosterol fed group is supported by the work of Kassem et al (2006) who reported that feeding diets containing 30% fenugreek seeds to female rabbits caused histopathologically evident proliferative changes of the endometrial glands and also a significant increase in the circulating plasma progesterone concentrations at 10 and 20 days of gestation.
Singh H, Chawla and Kapoor (1985) implied that a compound; 5-stigmastene-3p\ 7
sitosterol in a ratio of 4:1 in 20 hypercholesterolemic individuals, Phytosterols have also been reported to have anti-tumour properties (Ling and Jones 1995). Beta-sitosterol and taraxasterol have demonstrated inhibition of colon and breast cancer development at various stages including tumorigenesis, tumor promotion, and induction of cell differentiation (Ovesna, Vachalkova, Horvathova 2004). Feeding a 1-2% phytosterol mixture, containing 56% sitosterol, 28% campesterol, 10% stigmasterol and 6% dihydrobrassicasterol by weight, for 22 days resulted in normalizing the hyperproliferation of colonocytes, a symptom that precedes colon cancer development. Sitosterol has been found to induce apoptosis (programmed cell death) when added to cultured prostate, breast and colon cancer cells (Awad and Fink 2000). A 39% reduction in the number of rats with methylnitrosourea-induced tumor and a 60% reduction in the number of tumors per rat was observed with 0.2% sitosterol feeding for 28 weeks (Raicht et al 1980). In a six-month study of 200 men with symptomatic benign prostatic hyperplasia, the noncancerous enlargement of the prostate, 60 mg/d of a beta-sitosterol preparation improved symptom scores, increased peak urinary flow and decreased post-void residual urine volume compared to the placebo (Berges et al 1995).
Besides the above data on B-sitosterol and its effect on human health, information on the effect of y- Sitosterol or ergo-5-en-3-ol on human health is scarce. Given this premise this study was an attempt to prove the anti-estrogenic effect of y- Sitosterol and ergo-5-en-3-ol valuable to human nutrition. From the point of health and nutrition, this information can be used post partum to regain the original size of the uterus. The data can also serve as the template that can be transpired into alternative pharmaceutical preparations for inducing antiestrogenic effect when required.
SOURCE OF BIOLOGICAL RESOURCE MATERIAL:
For the work related to the current patent, the fenugreek seeds (variety: RMT-1) were specifically procured from the Agriculture Produce Market Committee (APMC), Navi Mumbai, Maharashtra. Approval for the same from the National Biodiversity Authority is under process.
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1. A therapeutic composition of a phytosterol extract isolated from fenugreek seeds comprising of y- Sitosterol and ergo-5-en-3-ol wherein the weight ratio of y- Sitosterol and ergo-5-en-3-ol is in the range of 81:19 to 85:15 for the antiestrogenic effect.
2. The composition of claim 1, wherein the anti-estrogen is y- Sitosterol and ergo-5-en-3-ol as a mixture.
6. DATE 19th JANVARY 2007
|Indian Patent Application Number||121/MUM/2007|
|PG Journal Number||07/2009|
|Date of Filing||19-Jan-2007|
|Name of Patentee||AMIN JYOTI|
|Applicant Address||C/201 AKRUTI ANERI, BEHIND MAROL MAROSHI BUS DEPOT, MAROL, ANDHERI (E), MUMBAI - 400 059, MAHARASHTRA, INDIA.|
|PCT International Classification Number||A61K38/16C, A61K36/48|
|PCT International Application Number||N/A|
|PCT International Filing date|